As used herein, the term "ligand-receptor assay" refers to an assay for at least one target ligand which may be detected by the formation of a complex between the ligand and a receptor capable of specific interaction with that target ligand. The target ligand may be the analyte itself or a substance which, if detected, can be used to infer the presence of the analyte in a sample. In the context of the present invention, the term "ligand", includes haptens, hormones, peptides, proteins, deoxyribonucleic acid (DNA), ribonucleic acids (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific ligand receptor therefor. Ligand-receptor assays are generally useful for the in vitro determination of the presence and concentration of ligands in body fluids, food products, animal fluids, and environmental samples. For example, the determination of specific hormones, peptides, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved the medical diagnosis of the human condition. There is a continuing need for simple, rapid assays for the qualitative, semi-quantitative, and quantitative determination of such ligands in a sample. Furthermore, in many situations, such assays need to be simple enough to be performed and interpreted by non-technical users.
Ligand-receptor assays rely on the binding of target ligands by ligand receptors to determine the concentrations of target ligands in a sample. Ligand-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize ligand receptors in substantial excess over the concentration of target ligand to be determined in the assay. Sandwich assays, in which the target ligand is detected by binding to two ligand receptors, one ligand receptor labeled to permit detection and a second ligand receptor, frequently bound to a solid phase, to facilitate separation from unbound reagents, such as unbound labeled first ligand receptor, are examples of non-competitive assays. Competitive assays generally involve a sample suspected of containing target ligand, a ligand analogue conjugate, and the competition of these species for a limited number of binding sites provided by the ligand receptor. Those skilled in the art will appreciate that many variations of this basic competitive situation have been previously described and will not be discussed in detail herein except where pertinent to the general objectives of this invention.
Competitive ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of target ligand is determined by its effect on the extent of binding between ligand receptor and ligand analogue conjugate. The signal observed is modulated by the extent of this binding and can be related to the amount of target ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive ligand-receptor assay in which the ligand analogue conjugate is a ligand analogue-enzyme conjugate and the ligand receptor is capable of binding to either the target ligand or the ligand analogue. The binding of the antibody to the ligand analogue-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound target ligand and ligand analogue-enzyme conjugate for ligand-receptor binding sites, as the target ligand concentration increases the amount of unbound ligand analogue-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
Heterogeneous, competitive ligand-receptor assays require a separation of ligand analogue conjugate bound to ligand receptor from the free ligand analogue conjugate and measurements of either the bound or the free fractions. Methods for performing such assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, 4,425,438, and 4,506,009, European Patent Application 87309724.0, and PCT International Application No. PCT/US86/00668. Separation of the bound from the free may be accomplished by removal of the ligand receptor and anything bound to it from the free ligand analogue conjugate by immobilization of the ligand receptor on a solid phase or precipitation. The amount of the ligand analogue conjugate in the bound or the free fraction can then be determined and related to the concentration of the target ligand in the sample. Normally the bound fraction is in a convenient form, for example, on a solid phase, so that it can be washed, if necessary, to remove remaining unbound ligand analogue conjugate and the measurement of the bound ligand analogue conjugate or related products is facilitated. The free fraction is normally in a liquid form that is generally inconvenient for measurements. If multiple ligands are being determined in a single assay, the determination of the free fraction of ligand analogue conjugate for each ligand is made impossible if all are mixed in a single liquid unless the responses of the individual ligand analogue conjugates can be distinguished in some manner. However, detecting the free fraction of ligand analogue conjugate in assays that are visually interpreted is a distinct advantage because the density of the color developed in such assays is generally proportional to the ligand concentration over much of the range of ligand concentration.
One method that can be used to detect the free ligand analogue conjugate in a heterogeneous, competitive ligand-receptor assay process is to provide a second, immobilized receptor specific for the target ligand on a solid phase so that the ligand analogue conjugate not bound to the first ligand receptor can be bound to the second ligand receptor immobilized on the solid phase. A serious problem with this approach is that the concentration of target ligand in the sample is often several orders of magnitude larger than the concentration of ligand analogue conjugate used in the assay process. Under these circumstances, the target ligand and the ligand analogue conjugate compete for the available binding sites on the first ligand receptor resulting in essentially all of the ligand analogue conjugate being free in the assay fluid. When the assay fluid is contacted with the immobilized second receptor, the free target ligand and the free ligand analogue conjugate compete for binding sites provided by the second ligand receptor. The excess of free target ligand is such that its concentration remains several orders of magnitude larger than that of the free ligand analogue conjugate so that the second ligand receptor binding sites on the solid phase are substantially filled by the target ligand. The result of this assay process is that little or no signal may be observed on the solid phase when the concentration of the target ligand in the sample is high when in fact the assay should be designed to produce the maximum response for such concentrations of target ligand.
In European Patent Application No. 87309724.0, a method is described where the sample suspected of containing the target ligand and a ligand analogue conjugate are contacted with a bibulous strip that contains immobilized ligand receptor. When sufficient target ligand is present in the sample, free ligand analogue conjugate travels beyond the first immobilized ligand receptor zone and contacts a situs where either ligand receptor or another receptor capable of binding the ligand analogue conjugate is immobilized. If the receptor at the situs is receptor for the target ligand, then the problem of competition in the presence of high concentrations of target ligand exists as described above. Methods are described where the receptor at the situs is a receptor that binds to a species other than the ligand analogue on the free ligand analogue conjugate so that high concentrations of free target ligand do not compete for binding sites at the situs. The use of such receptors at the situs requires the development of additional ligand-receptor pairs for ligands unrelated to the target ligand for each target ligand to be assayed and restricts these assays to formats where the target ligand receptor is immobilized on a solid phase. Under these circumstances the assay of multiple target ligands in a single assay becomes complex and difficult to develop.
The method described in U.S. Pat. No. 4,506,009 utilizes a ligand analogue conjugate which has both the ligand analogue and an insolubilizing binding component coupled to the signal development element. An insolubilizing receptor is used to precipitate the free ligand analogue conjugate unless it is sterically hindered by the binding of the antibody specific for target ligand to the ligand analogue. This method overcomes some of the deficiencies of the prior art because it provides a method to determine the free fraction of ligand analogue conjugate without interference from the free target ligand, but it requires the coupling of two elements, the ligand analogue and the insolubilizing binding component, to the signal development element in such a way that the binding of the antibody to the ligand analogue sterically prevents the binding of the insolubilizing receptor to the insolubilizing binding component. The relative and absolute amounts of the ligand analogue and the insolubilizing binding component that are coupled to the signal development element must be empirically selected to achieve the desired result. The need for such manipulation is both time consuming and may limit the assay performance by restricting the ratio of ligand analogue per signal development element.
The present invention provides a method for the determination of the free fraction of ligand analogue conjugate in competitive ligand-receptor assays by utilizing antibodies that are specific for ligand analogue, the form of the ligand that is coupled to the signal development element. Such antibodies bind to the ligand analogue conjugate with substantially greater affinity than their affinity for the target ligand. We have discovered that such antibodies are produced as a result of the normal immune response to a ligand analogue coupled to an immunogenic carrier protein. Because no additional elements are incorporated into the ligand analogue conjugate and the selection of ligand analogue antibodies does not require additional antibody generation beyond that required to generate antibodies to the target ligand, the selection and utilization of ligand analogue antibodies provides a simple and effective means for the detection of free ligand analogue conjugate in competitive ligand-receptor assays, even in the presence of high concentrations of target ligand.